Thymus vulgaris L. essential oil and its main component thymol: Anthelmintic effects against Haemonchus contortus from sheep.

Haemonchus contortus is an important gastrointestinal parasite on sheep farms in tropical regions. The resistance of the parasite against most anthelmintic drugs represents a great economic problem to sheep farming and is a major challenge that needs to be overcome. The searches for new anthelmintic agents that act on different stages of the parasite's life cycle are necessary for the development of new therapeutic options. The aim of this study was to evaluate the in vitro and in vivo anthelmintic activity of Thymus vulgaris essential oil against H. contortus and of its main component, the monoterpene thymol. Despite the relative ineffectiveness of the oil in the in vivo test, which may be corrected in the future after technical improvements to increase the oil's bioavailability, the in vitro results validated the popular use of T. vulgaris oil as an anthelmintic agent, at least against H. contortus. In fact, both the essential oil and thymol, which accounts for 50.22% of the oil composition, were effective against the three main stages of H. contortus. The oil and thymol were able to inhibit egg hatching by 96.4-100%, larval development by 90.8-100%, and larval motility by 97-100%. Similar to the positive control (levamisole 20mg/mL), the oil and thymol completely inhibited the motility of H. contortus adults within the first 8h of the experiment. Since thymol reproduces the anthelmintic effects of the oil and because it is the main component of the oil, it is reasonable to assume that thymol is the most important compound responsible for the anthelmintic effect of T. vulgaris. These results are of ethnopharmacological importance and may contribute to the development of new drugs and even herbal medicines, increasing treatment options for the farm breeding.

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae) / Atividade proteolítica de produtos de excreção/secreção de Cochliomyia hominivorax (Diptera: Calliphoridae)

The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30µg, 40µg and 50µg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)

Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30µg, 40µg e 50µg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases, fato que pode estar relacionado a processos vitais para a sobrevivência do parasita, tais como a penetração nos tecidos dos hospedeiros e nutrição durante os estágios larvais.(AU)

Acaricidal action of destruxins produced by a marine-derived Beauveria felina on the bovine tick Rhipicephalus (Boophilus) microplus.

The increasing resistance of Rhipicephalus (Boophilus) microplus tick to commercial insecticides requires alternative methods for the control of this cattle plague. The enthomopathogenic fungus Beauveria felina produces destruxins in culture media, cyclic depsipeptides which display an array of biological activities. The present investigation aimed to evaluate the acaricide action of destruxins isolated from B. felina culture media on R. (B.) microplus engorged females. B. felina was grown in MF medium under 19 different growth conditions. HPLC-PDA analysis of chromatographic fractions obtained from the 19 different growth media extracts indicated the presence of destruxins in all lipophylic fractions. Such fractions were combined and subjected to separation by HPLC. Fractions containing distinct destruxins composition were tested against R. (B.) microplus. Two fractions, composed of destruxin Ed(1) and pseudodestruxin B and/or pseudodestruxin C (fraction P1) as well as by hydroxyhomodestruxin B and/or destruxin D and/or roseotoxin C (fraction P7), displayed 30% and 28.7% acaricidal efficacy, respectively. This activity profile in such low concentration is adequate to consider destruxins as potential leading compounds to be developed for tick biological control.

[Ovicidal and larvicidal activity in vitro of Eucalyptus globulus essential oils on Haemonchus contortus]. / Atividade ovicida e larvicida in vitro do óleo essencial de Eucalyptus globulus sobre Haemonchus contortus.

The objective of this work was to evaluate ovicidal and larvicidal effects of Eucalyptus globulus essential oil (EGEO) on Haemonchus contortus. The chemical composition determination of EGEO was through gas chromatography and mass spectrometry. Egg hatch test (EHT) was performed in concentrations 21.75; 17.4; 8.7; 5.43 e 2.71 mg x mL(-1). In larval development test (LDT) were used the concentrations 43.5; 21.75; 10.87; 5.43 e 2.71 mg x mL(-1). Each trial was conducted by negative control with Tween 80 (3%) and positive control, 0.02 mg x mL(-1) of thiabendazole in EHT and 0.008 mg x mL(-1) of ivermectin in LDT. The maximum effectiveness of EGEO on eggs was 99.3% in concentration of 21.75 mg x mL(-1) and on larvae was 98.7% in concentration 43.5 mg x mL(-1). The concentration of EGEO that inhibits 50% of the eggs and larvae was 8.3 and 6.92 mg x mL(-1), respectively. The oil chemical analysis identified as main component the monoterpen 1,8-cineol. EGEO presented ovicidal and larvicidal activities in vitro, revealing a good potential for use in the control of sheep and goat gastrointestinal nematodes.

Atividade ovicida e larvicida in vitro do óleo essencial de Eucalyptus globulus sobre Haemonchus contortus / Ovicidal and larvicidal activity in vitro of Eucalyptus globulus essential oils on Haemonchus contortus

O objetivo deste trabalho foi avaliar o efeito do óleo essencial de Eucalyptus globulus (OEEG) sobre a eclosão e desenvolvimento de larvas de Haemonchus contortus. A determinação da composição química do OEEG foi feita por meio de cromatografia gasosa acoplada à espectrometria de massa. No teste de eclosão de ovos (TEO), utilizaram-se as concentrações 21,75; 17,4; 8,7; 5,43 e 2,71 mg.mL-1; e no de desenvolvimento larvar (TDL) as concentrações foram 43,5; 21,75; 10,87; 5,43 e 2,71 mg.mL-1. Cada ensaio foi acompanhado por um controle positivo, 0,02 mg.mL-1 tiabendazol, no TEO e 0,008 mg.mL-1 ivermectina no TDL, e por um controle negativo com o diluente Tween 80 (3 por cento). A eficácia máxima obtida pelo OEEG sobre ovos foi de 99,3 por cento na concentração de 21,75 mg.mL-1 e, sobre larvas, 98,7 por cento na concentração de 43,5 mg.mL-1. A concentração do OEEG, que inviabilizou 50 por cento dos ovos e das larvas do parasito, foi de 8,3 e 6,92 mg.mL-1, respectivamente. A análise química do óleo identificou como principal componente o monoterpeno 1,8-cineol. O OEEG apresentou atividade ovicida e larvicida in vitro sobre H. contortus, determinando um bom potencial para utilização no controle de nematóides gastrintestinais de ovinos e caprinos.

The objective of this work was to evaluate ovicidal and larvicidal effects of Eucalyptus globulus essential oil (EGEO) on Haemonchus contortus. The chemical composition determination of EGEO was through gas chromatography and mass spectrometry. Egg hatch test (EHT) was performed in concentrations 21.75; 17.4; 8.7; 5.43 e 2.71 mg.mL-1. In larval development test (LDT) were used the concentrations 43.5; 21.75; 10.87; 5.43 e 2.71 mg.mL-1. Each trial was conducted by negative control with Tween 80 (3 percent) and positive control, 0.02 mg.mL -1 of thiabendazole in EHT and 0.008 mg.mL-1 of ivermectin in LDT. The maximum effectiveness of EGEO on eggs was 99.3 percent in concentration of 21.75 mg.mL-1 and on larvae was 98.7 percent in concentration 43.5 mg.mL-1. The concentration of EGEO that inhibits 50 percent of the eggs and larvae was 8.3 and 6.92 mg.mL-1, respectively. The oil chemical analysis identified as main component the monoterpen 1,8-cineol. EGEO presented ovicidal and larvicidal activities in vitro, revealing a good potential for use in the control of sheep and goat gastrointestinal nematodes.